RNF8-FHA domain-modified protein and method of producing the same

Abstract

Provided is an antigen-binding protein prepared merely by a method of in vitro selection using the RNF8-FHA domain, which has no intramolecular disulfide bond and functions in cells as it is. One to four loops extending from the FHA domain are randomized, and a recognition site for a target molecule is artificially created on the FHA domain surface to construct an RNF8-FHA domain library. Using the library, an antigen-binding protein is efficiently selected in vitro.

The invention claimed is: 
     
       1. A method of producing an artificial antibody against a predetermined antigen, comprising:
 (i) preparing a RNF8-FHA domain library comprising randomized-loop polypeptides obtained by randomizing two or more of the following loops (a′) to (d′) in the E3 ubiquitin ligase RNF8-FHA domain comprising the amino acid sequence of SEQ ID NO:1: 
 (a′) a loop consisting of the 41 st -46 th  amino acid residues of the RNF8 protein shown by SEQ ID NO:1, 
 (b′) a loop consisting of the 49 th -62 nd  amino acid residues of the RNF8 protein shown by SEQ ID NO:1, 
 (c′) a loop consisting of the 78 th -83 rd  amino acid residues of the RNF8 protein shown by SEQ ID NO:1, 
 (d′) a loop consisting of the 108 th -118 th  amino acid residues of the RNF8 protein shown by SEQ ID NO:1, 
 wherein each of the randomized-loop polypeptides comprises the amino acid sequence shown by SEQ ID NO:1 except that a random sequence is introduced into two or more loops selected from the group consisting of (a′) to (d′), and no amino acid residues are deleted from, substituted for, inserted into, or added to any regions of the amino acid sequence other than loops (a′), (b′), (c′), and (d′), and 
 (ii) selecting a randomized-loop polypeptide that binds to the antigen as the artificial antibody from among the randomized-loop polypeptides of the RNF8-FHA domain polypeptide library, 
 wherein unmodified RNF8-FHA domain has substantially no binding affinity for the antigen. 
 
     
     
       2. The method according to  claim 1 , wherein the number of amino acid residues in the random sequence is 3-12 residues. 
     
     
       3. The method according to  claim 2 , wherein selecting a randomized-loop polypeptide that binds to the antigen from the randomized-loop polypeptides is performed by in vitro selection. 
     
     
       4. The method according to  claim 1 , wherein selecting a randomized-loop polypeptide that binds to the antigen from the randomized-loop polypeptides is performed by in vitro selection. 
     
     
       5. The method according to  claim 1 , wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing a region in each of the loops with the random sequence, wherein the region and the random sequence have the same number of amino acid residues. 
     
     
       6. The method according to  claim 1 , wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing each of the loops with the random sequence. 
     
     
       7. The method according to  claim 2 , wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing a region in each of the loops with the random sequence, wherein the region and the random sequence have the same number of amino acid residues. 
     
     
       8. The method according to  claim 2 , wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing each of the loops with the random sequence.